Summary/Abstract While vaccine-preventable, chronic infection with hepatitis B virus (HBV) remains a global public health problem: HBV-induced liver cirrhosis and liver cancer kill about 2,000 people in the United States and roughly 620,000 people worldwide each year. Globally, WHO reported 257 million people, or 3.5% of the population, were living with chronic HBV infection in the world in 2015. Untreated chronic viral hepatitis can progress to life-threatening complications, such as cirrhosis or hepatocellular carcinoma. Coinfections with other viruses, such as HIV, are also common. Once affected individuals are diagnosed, patients are placed on long-term treatment. The goals of HBV therapy are to stop or reverse liver inflammation and fibrosis, through sustained suppression of HBV replication. However, to be effective, therapies require continuous monitoring, since resistance to treatment can develop, especially in non-compliant patients. While immunoassays were the norm for diagnosing HBV patients, nucleic acid amplification tests (NAATs) have become standard of care for HBV. Since viral load (VL) testing is the most important indicator of response to therapy, VL should be measured in all patients at baseline and then on a regular basis, especially in patients on treatment. VL is usually measured with sophisticated instruments, and there currently are no methods for monitoring HBV viral load that combine speed, accuracy, low cost, and the option for Point-Of- Care. A truly POC NAAT for HBV that provided viral load information would have an immediate clinical impact. We propose to develop an accurate, rapid, and inexpensive paper-based molecular test for HBV Viral Load semi-quantification that is as accurate as gold standard NAATs but far less expensive, and that can be implemented at the POC. We have developed a paperfluidic molecular diagnostic platform that performs well with several isothermal nucleic acid amplification assays. We will combine innovations from this platform with a novel, tunable semi- quantitative assay based on predictable stratification of target amplicons based on their abundance. In this developmental program we will focus on development and optimization of the assay, and the initial steps to make the system portable, setting the stage for full integration in future work. This work has the potential to change how we manage care for chronic HBV patients.